Artificial Vitreous Humor for the Investigation of Drugs and Drug Formulations

ABSTRACT

The invention relates to an artificial vitreous humor composition comprising a phosphate buffer, wherein the phosphate buffer has a pH value in the range from 7.0 to 7.7, particularly from 7.1 to 7.6, more particularly from 7.2 to 7.5. The invention further relates to a method of production of an artificial vitreous humor composition, a method for analyzing the behavior of a substance, a method for analyzing the change of the artificial vitreous humor composition upon contact with a substance and a kit of parts.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/EP2018/078937, filed Oct. 23, 2018, which is claims benefit ofpriority to European Patent Application No. 17198210.1, filed Oct. 25,2017, which are incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to artificial vitreous humor compositions, amethod for the production of an artificial humor, a method for analyzingthe behavior of a substance and a kit of parts.

BACKGROUND

For the treatment of macular degeneration or retina diseases such as forexample diabetic retinopathy, intravitreal injections of drugs haveproven successful. In intravitreal injections a drug formulation isdirectly injected into the vitreous humor (VH), that is, the clear gelfilling the space between lens and retina of the eyeball of for examplehumans. The fraction of an administered dose of an unchanged drug thatreaches the retina is high, thus intravitreal injections usually havehigh bioavailability.

However, in-vitro tests to study for example stability of a drugformulation in VH have proven little useful when simulating real in vivoconditions. VH, when not in its natural environment, degenerates fastand the pH value of VH may increase rapidly due to accumulation ofdegenerated products in the VH. Thus, tests may not represent the actualsituation in an eye of a living subject, especially not over longperiods of time, e.g. several days. In more advanced test systems, thephysiological pH value of VH may be stabilized by applying a bufferingsystem. Therein, degradation products are allowed to leave the VHthrough a semi-permeable membrane into a buffer solution.

Following intravitreal (IVT) injection of a drug formulation or adrug-loaded long acting delivery (DDS) system (E. P. Holowka, S. K.Bhatia, Controlled release systems, Drug Delivery: Materials Design andClinical Perspective, Chapter-2, Springer Science Business Media, NewYork, 2014, ISBN: 978-1-4939-1997-0, 7-62.), the therapeutic agentremains in the VH and gets exposed at physiological pH and temperaturefor significantly longer duration. Therefore, it is of prime interest tostudy the stability of these agents in VH at physiological conditions.Physical and chemical variability of the natural human VH is accountedfrom disease condition and age, which may have direct impact on thestability predictions. When VH of animal origin is used (e.g. rabbit,pig, monkey or others), there are significant biological intra- andinter-species variabilities e.g. relating to variabilities in viscosityand/or density.

It is known that the natural VH is composed of various small and largeinorganic and organic molecules which can be largely categorized asbuffer system, small molecular weight components (e.g. salts and ions)and matrix forming polymers (e.g. collagen and hyaluronic acid). Indetail natural VH is largely composed of water (98-99%). The remainingconstituents (1-2%) are mainly collagen, hyaluronic acid, proteins andsmall mollecular weight components such as ascorbic acid, glutathione,glucose, lactate, xanthine, hypoxanthine, creatinine and urea asdisclosed in Kleinberg et al. (Vitreous substitutes: a comprehensivereview, Sury Ophthalmol. 2011 July-August; 56(4): 300-23) and AmithMulla (Role of vitreous humor biochemistry in forensic pathology,University of Saskatchewan, Thesis, 2005).

There is a need to solve the aforementioned challenges by developing anartificial vitreous humor (aVH), which is able to offer better controlover the physical and chemical parameters, and improves robustness ofthe investigation. This is achieved by the features of the independentclaims.

SUMMARY OF THE INVENTION

We have evaluated novel compositions of aVH comprising a buffer system,small molecular weight (SMW) components and large molecular weight (LMW)components such as matrix forming polymers, which can then serve as anin-vitro model for the prediction of drug or drug formulation stability,and drug release behavior from DDS systems following IVT delivery. Thenovel aVH would prove as the simplest yet very efficient real-timein-vitro experimental medium for stability and release prediction.Hence, the novel aVH compositions as described herein may serve asresearch tool but also as quality control tool.

According to a first aspect of the invention, an artificial vitreoushumor composition is provided comprising a phosphate buffer. Thephosphate buffer has a pH value in the range from 7.0 to 7.7,particularly from 7.1 to 7.6, more particularly from 7.2 to 7.5. Mostparticularly the phosphate buffer has a pH value of 7.4. The phosphatebuffer may be selected from the group consisting of phosphate buffersaline (PBS), phosphate buffer saline including Tween (PBST), phosphatebuffer saline including Tween and sodium azide (PBSTN) and phosphatebuffer saline including sodium azide (PBSN).

The phosphate buffer may be phosphate buffer saline (PBS). The phosphatebuffer saline may be in the range particularly from 0.001 to 0.2 M, moreparticularly from 0.003 to 0.05, most particularly from 0.005 to 0.02 M.The phosphate buffer saline preferably has a pH value of 7.4. Apreferred buffer according to the invention is 0.01 M PBS, pH 7.4comprising 8 gm/L NaCl, 0.2 gm/L KCl, 1.44 gm/L Na₂HPO₄ and 0.24 gm/LKH₂PO₄.

The artificial vitreous humor composition may comprise at least onesmall molecular weight component. The small molecular weight componentmay be selected from the group consisting of creatinine, glucose, urea,xanthine, hypoxanthine, sodium lactate and glutathione. The artificialvitreous humor composition may comprise at least one of up to 578 μMcreatinine, up to 28.1 mM glucose, up to 58.5 mM urea, 200 to 1630 μMxanthine, 100 to 800 μM hypoxanthine, 0.1 to 23 mM sodium lactate, 50 to300 μM glutathione. In a preferred embodiment, the artificial vitreoushumor composition comprises 64.6 μM creatinine, 2.2 mM glucose, 7.6 mMurea, 580 μM xanthine, 309 μM hypoxanthine, 10.5 mM sodium lactate and200 μM glutathione.

Furthermore, the artificial vitreous humor composition may comprise 20to 300 mg/L type II collagen and/or 0.03 to 0.9% w/v sodium hyaluronate.Collagen and/or sodium hyaluronate serve as matrix forming components inthe artificial vitreous humor. In a preferred embodiment of theinvention the artificial vitreous humor compositions comprises 40 mg/Ltype II collagen and/or 0.6% w/v sodium hyaluronate.

The artificial vitreous humor composition may not comprise ascorbicacid.

A further aspect of the invention relates to a method for the productionof an artificial vitreous humor composition, preferably as describedherein. The method comprises step (i), wherein stock solutions ofglucose, creatinine, sodium lactate, glutathione and urea in water, astock solution of hypoxanthine in formic acid:water of 2:1 ratio, and astock solution of xanthine sodium in 1M NaOH are provided. The methodfurther comprises step (ii) of mixing of the stock solutions of step (i)in a phosphate buffer with a pH value in the range from 7.0 to 7.7,particularly from 7.1 to 7.6, more particularly from 7.2 to 7.5, mostparticularly a pH value of 7.4, and pH adjustment to the pH value of thephosphate buffer. Preferably, mixing is performed with constant stirringof the mixture. In step (iii) hyaluronic acid is added to the mixtureobtained in step (iii) and the mixture is stirred at 2 to 8° C. for atleast 4 hours. Particularly, the mixture is stirred until the hyaluronicacid is completely dissolved. The method further comprises a step (iv),wherein collagen type-II is added, preferably with constant stirring, tothe mixture obtained in step (iii). The mixture obtained in step (iv) isoptionally sterile filtered.

The phosphate buffer may be selected from the group consisting ofphosphate buffer saline (PBS), phosphate buffer saline including Tween(PBST), phosphate buffer saline including Tween and sodium azide (PBSTN)and phosphate buffer saline including sodium azide (PBSN). The phosphatebuffer may be phosphate buffer saline (PBS). The phosphate buffer salinemay be in the range particularly from 0.001 to 0.2 M, more particularlyfrom 0.003 to 0.05, most particularly from 0.005 to 0.02 M. Thephosphate buffer saline preferably has a pH value of 7.4. A preferredbuffer according to the invention is 0.01 M PBS, pH 7.4 comprising 8gm/L NaCl, 0.2 gm/L KCl, 1.44 gm/L Na₂HPO₄ and 0.24 gm/L KH₂PO₄.

The artificial vitreous humor composition may comprise at least onesmall molecular weight component. The small molecular weight componentmay be selected from the group consisting of creatinine, glucose, urea,xanthine, hypoxanthine, sodium lactate and glutathione. In a preferredembodiment, the aVH composition comprises 64.6 μM creatinine, 2.2 mMglucose, 7.6 mM urea, 580 μM xanthine, 309 μM hypoxanthine, 10.5 mMsodium lactate and 200 μM glutathione. Furthermore, the artificialvitreous humor composition may comprise 20 to 300 mg/L type II collagenand/or 0.03 to 0.9% w/v sodium hyaluronate. Collagen and/or sodiumhyaluronate serve as matrix forming components in the artificialvitreous humor. In a preferred embodiment of the invention theartificial vitreous humor compositions comprises 40 mg/L type IIcollagen and/or 0.6% w/v sodium hyaluronate. In a preferred embodimentof the invention the artificial vitreous humor compositions comprises 40mg/L type II collagen and 0.6% w/v sodium hyaluronate. The artificialvitreous humor composition may not comprise ascorbic acid.

A further aspect of the invention relates to a method for analyzing thebehavior of a substance applied to an artificial vitreous humorcomposition, preferably as described herein. The method comprises step(i), wherein an artificial vitreous humor composition, preferably asdescribed herein, is provided in an in-vitro environment. An in-vitroenvironment according to the invention may be a vial, preferably a glassvial or plastic vial, well, container, depreshion dish, ex-vivointravitreal (ExVit) model as described in Patel et al., Evaluation ofprotein drug stability with vitreous humor in a novel ex-vivointraocular model, European Journal of Pharmaceutics andBiopharmaceutics. 2017; 112:117-186 (which is incorporated herein byreference in its entirety), and any specially designed glass or plasticdevice. The method further comprises step (ii), wherein the substance tobe analyzed is applied to the artificial vitreous humor. The substancemay be administered to the artificial vitreous humor composition.Particularly, the substance may be injected to the artificial vitreoushumor composition or diffused into the artificial vitreous humorcomposition or released from a long-acting sustained release delivery(DDS) system into the artificial vitreous humor composition.Furthermore, the method comprises step (iii) wherein at least oneproperty of the applied substance is determined. As the property of thesubstance to be applied is known prior to the application to thevitreous humor, a comparison of the property of the substance before andafter application to the artificial vitreous humor is feasible; e.g. inorder to analyze the stability of the applied substance when present inthe aVH, bioavailability, release from DDS system and degradation of theDDS system (Blanco et al., Protein encapsulation and release frompoly(lactide-co-glycolide) microspheres: effect of the protein andpolymer properties and of the co-encapsulation of surfactants, Europeanjournal of pharmaceutics and biopharmaceutics. 1998 May; 45(3): 285-94)within the aVH. The property of the substance may be determinedimmediately and/or in intervals in order to follow the change of theproperty over time.

The substance to be applied may be at least one of a macromolecule, aprotein, e.g. an antibody, a peptide, an oligonucleotide, an aptamer, adrug formulation, an excipient, a small molecule, a drug delivery system(biodegradable and nonbiodegradable), or a combination thereof. However,the term substance is not to be understood as being limited to theaforementioned examples.

The substance to be applied may comprise molecules having a size in arange between 100 Da and 1800 kDa, particularly in a range between 1 kDaand 500 kDa, more particularly between 4 kDa and 200 kDa, mostparticularly between 10 kDa and 175 kDa.

The at least one property of the applied substance is selected from thegroup consisting of stability, bioavailability, release from DDS systemand degradation of DDS system.

The applied substance may be left in the artificial vitreous humorcomposition for up to 360 days, particularly up to 180 days, moreparticularly up to 90 days, prior to step (iii). However, as mentionedearlier the property of the substance may be determined in intervals inorder to follow the change of the property with time. Then, step (iii)may define the last determination in time of the at least one property.The artificial vitreous humor may be maintained at a constanttemperature prior to step (iii), particularly at a temperature in therange from 32 to 38° C., more particularly from 35 to 37° C., mostparticularly from 36 to 37° C.

A further aspect of the invention relates to a method for analyzing thebehavior of an artificial humor composition as described herein, whereina substance is applied to the artificial humor composition. The methodcomprises step (i), wherein an artificial vitreous humor composition,preferably as described herein, is provided in an in-vitro environment.An in-vitro environment according to the invention may be a vial,preferably a glass vial or plastic vial, well, container, depreshiondish, ex-vivo intravitreal (ExVit) model as described in Patel et al.,Evaluation of protein drug stability with vitreous humor in a novelex-vivo intraocular model, European Journal of Pharmaceutics andBiopharmaceutics. 2017; 112:117-186 (which is incorporated herein byreference in its entirety), and any specially designed glass or plasticdevice. The method further comprises step (ii), wherein a substance isapplied to the artificial vitreous humor. The substance may beadministered to the artificial vitreous humor composition. Particularly,the substance may be injected to the artificial vitreous humorcomposition or diffused into the artificial vitreous humor compositionor released from a long-acting sustained release delivery (DDS) systeminto the artificial vitreous humor composition. Furthermore, the methodcomprises step (iii) wherein at least one property of the artificialhumor composition is determined. As the property of the artificial humorcomposition is known prior to the application of the substance, acomparison of the property of the the artificial humor compositionbefore and after application of the substance is feasible; e.g. in orderto analyze the stability and/or viscosity of the the artificial humorcomposition when the substance is present. Generally, the method allowsan assessment of the tolerability of the artificial humor composition inview of the applied substance, particularly by determining the change ofat least one of the group consisting of viscosity, turbidity,osmolality. The property of the the artificial humor composition may bedetermined immediately and/or in intervals in order to follow the changeof the property over time. The substance to be applied may be at leastone of a macromolecule, a protein, e.g. an antibody, a peptide, anoligonucleotide, an aptamer, a drug formulation, an excipient, a smallmolecule, a drug delivery system (biodegradable and nonbiodegradable),or a combination thereof. However, the term substance is not to beunderstood as being limited to the aforementioned examples. Thesubstance to be applied may comprise molecules having a size in a rangebetween 100 Da and 1800 kDa, particularly in a range between 1 kDa and500 kDa, more particularly between 4 kDa and 200 kDa, most particularlybetween 10 kDa and 175 kDa. The applied substance may be left in theartificial vitreous humor composition for up to 360 days, particularlyup to 180 days, more particularly up to 90 days, prior to step (iii).However, as mentioned earlier the property of the the artificial humorcomposition may be determined in intervals in order to follow the changeof the property with time. Then, step (iii) may define the lastdetermination in time of the at least one property. The artificialvitreous humor may be maintained at a constant temperature prior to step(iii), particularly at a temperature in the range from 32 to 38° C.,more particularly from 35 to 37° C., most particularly from 36 to 37° C.

A further aspect of the invention relates to a method for analyzing thebehavior of a substance in a artificial humor as described herein and/orthe behavior of an artificial humor composition as described herein.

A further aspect of the invention relates to an artificial vitreoushumor composition as described herein produced by the method ofproduction as described herein.

A further aspect of the invention relates to a kit of parts for theproduction of an artificial vitreous humor composition, particularly asdescribed herein. The kit of parts comprises several components.Component (A) is a phosphate buffer, particularly phosphate buffersaline (PBS), more particularly 0.01 M phosphate buffer saline (PBS),having a pH value in the range from 7.0 to 7.7, particularly from 7.1 to7.6, more particularly from 7.2 to 7.5. most particularly a pH value of7.4. Component (B) comprises at least one selected from the groupconsisting of creatinine, glucose, urea, xanthine, hypoxanthine, sodiumlactate, glutathione. Preferably, component (B) comprises a mixture ofcreatinine, glucose, urea, xanthine, hypoxanthine, sodium lactate,glutathione or component (B) is a selection of components referred to as(B1) creatinine, (B2) glucose, (B3) urea, (B4) xanthine, (B5)hypoxanthine, (B6) sodium lactate and (B7) glutathione. Component (C)comprises type II collagen and/or sodium hyaluronate. Preferably,component (C) is a selection of components referred to as (C1) type IIcollagen and (C2) sodium hyaluronate. Optional component (D) isinstructions for use.

A further aspect of the invention relates to the use of an artificialvitreous humor composition as described herein in a method for analyzingthe behavior of a substance in the artificial humor composition asdescribed herein. A further aspect of the invention relates to the useof an artificial vitreous humor composition as described herein in amethod for analyzing the behavior of the artificial vitreous humorcomposition, when a substance is applied to the artificial vitreoushumor composition. A further aspect of the invention relates to the useof an artificial humor composition as described herein in a method foranalyzing the behavior of a substance in the artificial composition asdescribed herein and/or in a method for analyzing the behavior of theartificial humor composition, wherein a substance is applied to theartificial humor composition, as described herein.

A further aspect of the invention relates to the use of a phosphatebuffer saline having a pH value in the range from 7.0 to 7.7,particularly from 7.1 to 7.6, more particularly from 7.2 to 7.5, mostparticularly a pH value of 7.4 for the production of an artificialvitreous humor composition as described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Absorbance analysis correlating to the amount of reducedascorbic acid in three different simulated buffer media (SBM) preparedat different buffer strength after 1 week of incubation at 37° C.

FIG. 2: Determination of the viscosity of isolated porcine vitreoushumor.

FIGS. 3a and 3 b: Pictorial representations of the ExVit model utilizedto compare protein stability in the natural VH vs artificial VH (FIG. 3a) and of vial based model used to investigate protein stability in theartificial VH (FIG. 3b ).

FIGS. 4a and 4 b: Analysis of pH (FIG. 4a ) and osmolality (FIG. 4b ) ofthe PBS and vitreous humor (aVH and pVH) in ExVit or vial model with orwithout a monoclonal antibody (mAb) after incubation at 37° C.(Experiments were performed in triplicates and results are reported inmean±std. dev).

FIG. 5: Analysis of turbidity of the PBS and vitreous humor (aVH andpVH) in ExVit or vial model with or without mAb after incubation at 37°C. (Experiments were performed in triplicates and results are reportedin mean±std. dev).

FIGS. 6a and 6 b: Physical stability of mAb in aVH (ExVit or vialmodel), pVH and PBS were investigated by SEC. FIG. 6a represents themain peak. FIG. 6b represents the higher molecular weight species (HMWS,%) (Experiments were performed in triplicates and results are reportedin mean±std. dev).

FIGS. 7a and 7 b: Physical stability of mAb in aVH (ExVit or vialmodel), pVH and PBS was investigated by CE-SDS-NGS. FIG. 7a representsthe main peak. FIG. 7b represents the deglycosylated variants (area %).

FIGS. 8a and 8 b: Binding affinity of mAb. FIG. 8a represents bindingaffinity to antigen-1. FIG. 8b represents the binding affinity toantigen-2 retrieved from stability samples following incubation in aVH(ExVit or vial model) and pVH.

FIG. 9: Cumulative Release (%) of Small Molecule (SM) activepharmaceutical ingredients (API) from various PLGA based LADintravitreal implants.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. DEFINITIONS

“Vitreous humor” as used in this application may be from natural orartificial sources. Natural vitreous humor may be from various animalspecies, for example porcine, bovine, canine, feline, from rabbits andnon-human primates or may be human. Porcine vitreous humor isabbreviated pVH herein. Natural vitreous humor may for example be gainedfrom eyes that have previously been removed.

Artificial vitreous humor preferably mimics human vitreous humor.Artificial vitreous humor may be prepared from various polymericmaterials, also called large molecular weight (LMW) components or matrixforming components, for example natural polymers such as but not limitedto hyaluronic acid, alginate, agar, chitosan, gelatin, xanthan gum,pectins, collagen, or for example synthetic polymers such as but notlimited to pluronic gel, polyvinyl alcohol, polyphosphazenes, anydimeric, trimeric or multimeric gelling polymers composed of PEG, PCL,PLA, PGA, PLGA, poly acrylamide, polyacrylic acid, as well ascombinations of these polymers at different concentrations. The amountof LMW components, particularly the hyaluronic acid, may be chosen toachieve a defined viscosity of the final aVH composition. Artificialvitreous humor may further comprise small molecular weight (SMW)components. These small molecular weight components may be selected fromthe group comprising nitrogenous organic acids, sugars, reducing agents,such as urea, purine bases or derivatives thereof, salts of lactic acidand antioxidants, such as glutathione.

Vitreous humor may also be a mixture of artificial and natural vitreoushumor or their components in different combinations.

A “buffer solution” is a solution preferably having or being able tomaintain the pH value of the system, for example between pH 5.5 and pH8.5, preferably between about pH 7.0 and about pH 7.6, more preferablyat pH 7.4. “Physiologically relevant buffer solution” is a solutionpreferably having or being able to maintain a pH value of the systembetween about pH 7.0 and about pH 7.6, more preferably at pH 7.2-7.4.Preferably, a buffer solution comprises salts. The buffer solution mayfor example be a phosphate buffered saline (PBS), a bicarbonate buffer,Ringer's bicarbonate buffer, Ringer's lactate buffer, simulated bodyfluids, other isotonic solutions, cell culture medias, and any otherphysiologically representative buffers.

PBS according to the invention may comprise 8 gm/L NaCl, 0.2 gm/L KCl,1.44 gm/L Na₂HPO₄ and 0.24 gm/L KH₂PO₄. PBS preferably has a pH value of7.4.

The term “behavior of a substance” relates to the change of a propertyof a substance when applied to the artificial vitreous humor or else tothe artificial humor composition according to the invention. The changeof property of a substance can be a) chemical or physical changes, b)change in its binding to a target, c) transformation from onepolymorphic form to another polymorphic form, d) change of physicalstate, e.g. liquid to gel. A substance can be a therapeutic agent or twoor more therapeutic agents, excipient, formulation (simple or sustainedrelease) or placebo (simple or placebo of sustained releaseformulation).The composition of artificial VH (aVH) allows simulating invivo physiological conditions of natural VH. For example, with injectionof a drug formulation into vitreous humor, the formulation's stabilityand release from DDS may be simulated, for human but also for animaleyes. For example, different diffusion and precipitation behavior may betested for, for example, different proteins in different eye conditions(corresponding to more or less degenerated eyes, by age or by disease)for posterior eye tissue. These conditions can be simulated and testedwith the aVH and the method for analyzing the behavior of a substanceaccording to the invention by varying the properties, e.g. compositionand/or density and/or viscosity, of the aVH. In addition, more realistictest results may be achieved, especially on stability and release of asubstance from DDS, for example a protein or a drug formulation, bytaking into account the physical and chemical environment present at theinjection location. Thus, artificial vitreous humor and the methodaccording to the invention allow to more realistically simulate thegeometry of a natural environment, preferably of an eye environment.

The term “behavior of an artificial humor composition” relates to thechange of a property of the artificial humor composition, when asubstance is applied to the artificial vitreous humor according to theinvention. The change of property of the artificial humor compositioncan particularly be chemical or physical changes, and/or change ofphysical state, e.g. gel to liquid.

II. EXAMPLES

Materials

Porcine eyes were acquired from a local slaughter-house located nearZurich, Switzerland. Custom made side-by-side diffusion chambers werepurchased from SES GmbH-Analytical Systems (Bechenheim, Germany) Adiffusion controlling membrane, with molecular weight cut-off (MWCO) of50 kDa (cat #131384), was procured from Spectrum lab (California, USA).Collagen (type-II) (cat #804001-sol) and sodium hyaluronate (cat #HA15M)were purchased from mdbioproducts and Lifecore Biomedical, respectively.L-ascorbic acid (cat #A5960), xanthine sodium salt (cat #X3627),hypoxanthine (cat #H9636), creatinine (cat #C4255), urea (cat #51457),glucose (cat #G8270), L-glutathione (cat #G4251) and sodium lactate (cat#71718) were procured from Sigma Aldrich, USA. Bispecific monoclonalantibody (mAb) was manufactured in a CHO cell line and provided by F.Hoffmann-La Roche AG, Basel. The mAb is a full length humanized mAbbased on the IgG-1 format with an approximate molecular weight of 150kDa. mAb was formulated in histidine buffer (20 mM, pH 6.0) containingsugar and surfactant to provide tonicity and stability, respectively.The small molecule with a MW<600 Dalton and LogP of 3.24 was provided byF. Hoffmann-La Roche AG, Basel. PLGA polymers were procured from Evonik,Parsippany, USA (RG756S PL(DL)GA 75:25 and RG753H PL(DL)GA 75:25). Inall the experiments related to the ExVit model, phosphate buffer saline(0.01 M PBS, pH 7.4) was used in the buffer-compartment. All otherreagents utilized in these studies were of analytical grade.

Example 1

Development of a Simulated Buffer Medium (SBM)

Natural VH is a liquid tissue mainly composed of water with smallamounts of organic and inorganic components including matrix formingpolymers. The composition of VH is reported in literature. In order toprepare artificial VH (aVH), firstly, it was important to identify abase buffer system which can support the physicochemical stability ofaVH. Secondly, it was important to prepare a simulated buffer medium(SBM) containing all the small molecular weight components of thenatural VH in a defined concentration as reported in literature. Atlast, SBM was prepared in the presence of matrix forming polymers i.e.,hyaluronic acid and collagen, inherent components of natural VH.Stability of the aVH was then evaluated by storing it at 37° C. for 3months.

In order to identify the right buffer system, two biologically relevantbuffer systems supporting physiological pH such as Kerbs Ringerbicarbonate buffer and phosphate buffer saline (PBS) were investigated.The compositions of buffer systems are given in Table 1 and 2.

TABLE 1 Composition of bicarbonate Buffer (Modified Krebs ringer buffer,pH 7.4) Components Concentration MgCl₂ 0.0538 gm/L KCl  0.876 gm/L NaCl  9.29 gm/L Na₂HPO₄    0.1 gm/L NaH₂PO₄   0.18 gm/L NaHCO₃   1.26 gm/LCaCl₂  0.232 gm/L

TABLE 2 Composition of phosphate buffer saline (PBS, 0.01M, pH 7.4)Components Concentration NaCl    8 gm/L KCl  0.2 gm/L Na2HPO4 1.44 gm/LKH2PO4 0.24 gm/L

The following small molecular weight (SMW) components, hypoxanthine (309μM), creatinine (64.6 μM), xanthine (580 μM), ascorbic acid (350 μg/mL),glucose (2.2 mM), urea (7.6 mM), sodium lactate (10.5 mM) andglutathione (200 μM) are reported in the literature as key components ofthe VH. Hence, they were utilized for the preparation of SBM. Asdescribed in the Table 3 and Table 4, various compositions of SBM(composition 1-17) were prepared with and without specific SMWcomponents dissolved in either bicarbonate or PBS based bufferingsystem.

TABLE 3 Impact of small MW components on the storage stability ofsimulated buffer medium (SBM) at 37° C. with Kerbs Ringer bicarobonatebased buffering system Ascorbic Glucose Urea Sodium HypoxanthineCreatinine Xanthine acid (350 (2.2 (7.6 lactate Glutathione ClarityClarity pH pH # (309 μM) (64.6 μM) (580 μM) μg/mL) mM) mM) (10.5 mM)(200 μM) 3 days 7 days Initial 7 days 1 + + + + + + + + Clear TurbidPhysiological Acidic 2 + + + + + + + Turbid Turbid Physiological Acidic3 + + + + + + + Clear Turbid Physiological Acidic 4 + + + + + + + ClearTurbid Physiological Acidic 5 + + + + + + + Clear Turbid PhysiologicalAcidic 6 + + + + + + + Clear Turbid Physiological Acidic 7 + + + + + + +Clear Turbid Physiological Acidic 8 + + + + + + + Clear TurbidPhysiological Acidic 9 + + + + + + + Clear Turbid Physiological Acidic

TABLE 4 Impact of SMW components on the storage stability of simulatedbuffer medium (SBM) at 37° C. with Phosphate Buffer Saline (PBS, pH 7.4,0.01M) based buffering system Ascorbic Glucose Urea Sodium HypoxanthineCreatinine Xanthine acid (350 (7.6 lactate Glutathione Clarity ClaritypH pH # (309 μM) (64.6 μM) (580 μM) μg/mL) (2.2 mM) mM) (10.5 mM) (200μM) 3 days 7 days Initial 7 days 10 + + + + + + + + Clear ClearPhysiological Acidic 11 + + + + + + + Clear Clear Physiological Acidic12 + + + + + + + Clear Clear Physiological Acidic 13 + + + + + + + ClearClear Physiological Acidic 14 + + + + + + + Clear Clear PhysiologicalAcidic 15 + + + + + + + Clear Clear Physiological Acidic16 + + + + + + + Clear Clear Physiological Acidic 17 + + + + + + + ClearClear Physiological Acidic

These SBMs were sterile filtered with 0.22μ filter under laminar airflow. SBMs were then investigated for storage stability by incubating at37° C. for 7 days. Buffer compatibility was evaluated by measuring pHand clarity. For pH estimation, samples (100 μL) were asepticallytransferred in eppendorf tubes and analyzed by calibrated pH meter (827pH Lab, Metrohm, Switzerland). Clarity of the SBMs was observed visuallyand the samples were classified as clear or turbid.

In Table 3, all the studied compositions (1-9) containing Kerbs Ringerbicarbonate buffer showed turbidity and instability with respect to pHafter one week of incubation. The instability with respect to pH relatesto an acidic shift. This may be due to the weak buffering capacity ofbicarbonate based buffer system and/or due to the incompatibility ofbuffer system with small molecular weight components. In summary, itwould not be possible to develop SBM with Kerbs Ringer bicarbonatebuffer mainly due to turbidity and pH shift.

On the other hand, PBS based SBM compositions 10-17 remained clear after7 days of incubation at 37° C. (Table 4). However, all of thesecompositions also showed acidic pH shift after 7 days.

Example 2

Impact of Higher Buffer Strength, Sodium Lactate, Hypoxanthine andAscorbic Acid on the Stability of SBMs

In order to find stable composition, SBM with or without sodium lactate,hypoxanthine and ascorbic acid, and SBM with higher buffer strength wereinvestigated. The SBMs were prepared as reported in Table 5.

TABLE 5 Impact of buffer strength and presence/absence of sodiumlactate, hypoxanthine, and ascorbic acid on the storage stability ofsimulated buffer medium (SBM) at 37° C. Ascorbic Glucose Urea Sodium PBSHypoxanthine Creatinine Xanthine acid (350 lactate Glutathione pH pH #(pH 7.4) (309 μM) (64.6 μM) (580 μM) μg/mL) (2.2 mM) (7.6 mM) (10.5 mM)(200 μM) Initial 7 days 10  0.01M + + + + + + + + Physiological Acidic18 0.015M + + + + + + + + Physiological Acidic 19  0.02M + + + + + + + +Physiological Acidic 20  0.04M + + + + + + + + Physiological Acidic 210.015M + + + + + + + Physiological Acidic 22  0.02M + + + + + + +Physiological Acidic 23 0.015M + + + + + + + Physiological Acidic 24 0.02M + + + + + + + Physiological Acidic 25  0.01M + + + + + +Physiological Acidic 26 0.015M + + + + + + Physiological Acidic 27 0.02M + + + + + + Physiological Acidic 28  0.01M + + + + + + +Physiological Stable 29  0.02M + + + + + + + Physiological Stable 30 0.04M + + + + + + + Physiological Stable

SBMs were sterile filtered and incubated at 37° C. for one week andanalyzed for pH and clarity. Samples were collected and analysis wasperformed as described above. Also, the amount of reduced ascorbic acidwas analyzed in the samples using 1,1-diphenyl-2-picrylhydrazyl(α,α-diphenyl-β-picrylhydrazyl (DPPH) assay. Briefly, 100 μL of ascorbicacid standard or test samples were incubated in presence of 1900 μL ofDPPH solution at 37° C. for 30 min in dark. Samples were then analyzedfor change in optical density (OD) at 517 nm against methanol as blank.The presence of ascorbic acid turns color of solution from violet tobrownish-pale yellow.

The pH results depicted in Table 5 clearly show that higher bufferstrength (compositions 18-20) was not sufficient to maintain the pH.Also, absence of sodium lactate and hypoxanthine or both also could notprevent the acidic pH shift (compositions 21-27). Only SBM compositions28-30, without ascorbic acid exhibited stable pH after 7 days.Furthermore as shown in the DPPH of FIG. 1, SBMs with or withoutAscorbic acid exhibited similar levels of absorbance at 517 nmsuggesting absence of reduced form of ascorbic acid after 7 days ofincubation at 37° C. Ascorbic acid might be oxidized in less than 7 daysresulting in the pH shift to the acidic side. Hence, ascorbic acid wasnot included in the final SBM composition.

Example 3

Long Term Stability of SBM

Once the composition 28 of SBMs was found stable for 7 days withoutchange in pH or clarity, it was further characterized for long-termstorage stability at 37° C. Briefly, composition 28 was prepared andaseptically filtered under LAF. The sterile SBM was then transferredaseptically in 6 mL sterile glass vials. The glass vials were sealed andincubated at 37° C. for 3 months. The samples were collected at varioustime points such as, initial, week-1, week-2, week-5, week-8 andweek-12, and analyzed for pH, clarity, density, viscosity andosmolality. For pH estimation, samples (100 μL) were asepticallytransferred in eppendorf tubes and analyzed by calibrated pH meter (827pH Lab, Metrohm, Switzerland). Clarity of the SBMs was observed visuallyand the samples were classified as clear or turbid. Density of the SBMwas estimated at 20° C. using DMA 38 (Anton Paar) densiometer.Osmolality of the samples were estimated with a calibrated osmometer(Osmomat 030 3P Cryoscopic Osmometer) in the range of 0-0.5 Osmol/kgaccording to European Pharmacopoeia, Osmolality, Section 2.2.35,European Directorate for the Quality of Medicine, Strasbourg, France,2017, pp. 59. Following calibration, 50 μL of the samples were placed inthe osmometer and the osmolality was evaluated. Viscosity was analyzedusing SV-1A vibro viscometer. Briefly, 2 mL of sample was transferred inthe sample holder and vibrating plates were inserted in the samplesmaking sure that plates do not touch at the bottom or side walls ofsample holder. Viscosity was measured and reported in mPa·S.

TABLE 6 Long-term storage stability of simulated buffer medium (SBM) at37° C. Week Week Week Week Week Initial 1 2 5 8 12 Density 1.006 — —1.0057 1.0057 1.0056 Osmolality 0.297 0.295 0.295 0.298 0.297 0.299(Osmol/kg) pH 7.27 7.31 7.31 7.32 7.29 7.31 Viscosity 1.00 1.02 0.991.03 1.01 1.02 (mPa · s) Clarity Clear Clear Clear Clear Clear Clear

The data depicted in Table 6 show that SBM remained stable during thestudy period of 3 months. No change in pH, clarity, density, osmolalityor viscosity were observed during the investigation. Hence, the SBMcomposition without ascorbic acid at lower PBS strength (composition-28)was selected as suitable SBM for the development of aVH.

Example 4

Viscosity Assessment of Natural Vitreous Humor

Viscosity of the study/release medium (in this case aVH) would havesignificant impact on the rate of release of therapeutic agents from theDDS systems. Therefore, viscosity of the aVH has to be similar to thenatural VH. As mentioned earlier, VH is the liquid tissue which behaveslike a gel under pressure when it is in the eye. As soon as VH isisolated from the eye, it liquefies mainly due to the disorientation ofcollagen fibers. Rheological properties of VH are highly influenced bycollagen fibrillary concentration and orientation. Usually collagenfibrils are arranged in a specific order initiating from vitreous baseto the back of the eye (Le Goff et al., Adult vitreous structure andpostnatal changes., Eye. 2008 October; 22(10): 1214-22). Hence, it isvery important to estimate the viscosity of the VH in-situ withoutdamaging structure of VH matrix. Due to the complexity in analyticalprocedure, no specific information about the actual in-vivo viscosity ofthe natural VH is available. Here, we have estimated the viscosity ofnatural VH in-situ using a vibro viscometer, so this information can beused to prepare aVH with appropriate viscosity.

Freshly isolated porcine eyes were procured from a slaughter housewithout any further processing such as heat treatment. Throughout thetransportation, the eyes were stored in isotonic solution kept in an icebath. Prior to the viscosity measurement, the eyes were stored at 37° C.for 2 hours. After the incubation, eyes were cut open from sclera (1×1cm) to access VH. Vibration plates were inserted in the eyes making surethat the plates do not touch at the bottom or side walls. Also, tounderstand the impact of orientation of collagen fiber on the overallviscosity, the viscosity was measured by placing vibrating platesperpendicular or parallel to collagen fibers.

As described in FIG. 2, natural, here porcine, VH exhibited 71±43 mPa·Sof viscosity when the plates were placed parallel to the collagenfibers. Interestingly, viscosity was not significantly different whenthe plates were located perpendicularly to the collagen fibers (78±42mPa·S). Because hyaluronic acid along with collagen (in a definedorientation) is mainly responsible for the viscosity of the natural VH,the same components were considered as a viscosity enhancer in the aVH.According to concentration vs viscosity results of hyaluronic acidsolution (Table 7), 0.6% w/v of HA was used to prepare aVH. Here, it isimportant to note that similar viscosity of the aVH can be achievedusing different molecular weight hyaluronic acids at differentconcentrations.

TABLE 7 Viscosity of aqueous hyaluronic acid solution with an averagemolecular weight of 1.01 to 1.8 MDa at different concentrationshyaluronic acid Viscosity (Concentration % w/v) (mPa · s) 0 0.98 ± 0  0.03 1.78 ± 0.02 0.1 7.47 ± 0.01 0.2 18.3 ± 0.13 0.4 42.24 ± 0.22  0.672.88 ± 2.88  0.9 139.37 ± 2.80  

Example 5

Preparation of a Preferred Artificial VH Using SBM

Once the long-term stability of SBM was ensured, it was utilized toprepare aVH. Briefly, optimized SBM was prepared as described inprevious section and then a defined amount of hyaluronic acid (HA) wasadded to prepare 0.6% of HA-SBM solution. HA was solubilized in SBM bystirring overnight at 2-8° C. The following day, collagen type-II wasadded in the HA-SBM solution to prepare the final collagen concentrationof 40 mg/L. The resulting aVH was aseptically filtered through 0.22μfilter. The aVH was stored at 2-8° C. until further use. The finalcomposition of a preferred aVH is reported in Table 8.

TABLE 8 Composition of a preferred artificial vitreous humor ComponentsConcentration Optimized PBS, pH 7.4 NaCl  6.4 gm/L Simulated KCl 0.16gm/L Buffer Medium Na₂HPO₄•2H₂O 1.80 mg/L (SBM) KH₂PO₄ 0.24 gm/L SmallMW Creatinine 64.6 μM components Glucose  2.2 mM Urea  7.6 mM Xanthine 580 μM Hypoxanthine  309 μM Sodium lactate 10.5 mM Glutathione  200 μMMatrix forming components Collagen (type II)   40 mg/L SodiumHyaluronate 0.6% w/v

Example 6

Storage Stability of Artificial Vitreous Humor (aVH)

aVH was investigated for the storage stability at 37° C. For this study,3 mL of aVH was aseptically transferred in 6 mL sterile glass (type-I)vial and incubated at 37° C. for 3 months. Samples were collected atpre-defined time intervals i.e., initial, week-1, week-2, week-5, week-8and week-12, and analyzed for pH, osmolality, density and viscosity. Theanalysis was performed with the protocols described herein.

TABLE 9 Long-term storage stability of artificial VH at 37° C. InitialWeek 1 Week 2 Week 5 Week 8 Week 12 Density 1.0073 — — 1.0073 1.00741.0074 Osmolality (Osmol/kg) 0.307 0.303 0.300 0.307 0.306 0.308 pH 7.157.19 7.18 7.19 7.19 7.19 Viscosity (mPa · s) 71.5 72.7 74.9 73.4 72.663-66

As described in Table 9, all the physicochemical parameters remainedstable throughout the study period. Only a minor change in the viscosityat 3 month time point was observed which may be due to the partialdegradation of the HA polymer. Hence, in a next step aVH was comparedwith natural VH for the evaluation of protein stability.

Once, the aVH was found stable for 3 months at 37° C., 50 μl of 120mg/ml bispecific monoclonal antibody (mAb) with a molecular weight ofapproximately 150 kDa was injected in the aVH and natural VH, andinvestigated for its stability. It was hypothesized that if thestability of mAb in natural VH and aVH found comparable, it can beconfirmed that aVH has the potential to substitute natural VH forin-vitro/ex-vivo stability and/or release (from DDS formulations)investigations.

Example 7

Stability of mAb in the aVH and Natural VH (Experimental Setup)

In Patel et al., (Evaluation of protein drug stability with vitreoushumor in a novel ex-vivo intraocular model. European Journal ofPharmaceutics and Biopharmaceutics. 2015; 95(Pt B):407-17) and Patel etal. (Evaluation of protein drug stability with vitreous humor in a novelex-vivo intraocular model. European Journal of Pharmaceutics andBiopharmaceutics. 2017; 112:117-186) it was reported that as soon as theVH is isolated from the eye, the pH of the VH increases rapidly (to ca.pH 8.5). The rationale for this alkaline pH shift is not wellunderstood, but the literature points towards the speedy alterations inthe microenvironment of the VH upon isolation and/or the accumulation ofdegradation products of the VH. The elevated pH value (ca. pH 8.5) andconcentrated degradants in the isolated VH can have a detrimental impacton the protein stability and can stimulate degradation of mAb which doesnot occur in in-vivo situation and make this study prone to artefacts.Presently, only one reliable tool developed is available to study thestability of protein drugs or drug delivery systems in the isolated pVH.The aforementioned ExVit model has successfully solved the problems withalkaline pH shift and generation of VH degradation products. In order toestablish aVH as a stability prediction tool, it is very important toperform comparative study between aVH (in ExVit and vials) and isolatednatural VH (in ExVit). Hence, three test groups were studied namely, 1)protein incubated in the aVH using the ExVit model, 2) protein incubatedin isolated porcine VH (pVH) using the ExVit model, and 3) proteinincubated in the aVH in a close vial model.

Isolation of the Porcine Vitreous Humor (pVH)

Porcine eyes were opened with the incision placed near the conjunctivausing a dissecting knife and the clear VH was collected with thedisposable syringe without a needle. The VH was then sterile-filteredthrough a 0.22 μm filter to ensure removal of any microbialcontamination and cellular debris. The VH was stored (in small aliquots)below −70° C. to avoid possible metabolic activity and degradation orchange in VH. Throughout the process of VH isolation, eye balls werekept in an ice-bath. All the experiments were performed according to theAssociation of Research in Vision and Ophthalmology (ARVO) statement forthe use of animals in ophthalmic and vision research.

Experimental Set-Up

The ExVit is a two compartment model, VH-compartment andbuffer-compartment. As demonstrated in FIG. 3 a, two compartments wereseparated by a diffusion controlling membrane with MWCO of 50 kDa. Thebuffer-compartment was always filled with sterile PBS (0.01 M, pH 7.4)whereas the VH-compartment was filled with the sterile pVH or sterileaVH. The devices were sealed with a sterile Teflon cap and incubatedovernight at 37° C. (MaxQ-4000 incubator, Thermo Scientific). Followingincubation, PBS from the buffer-compartment was replaced with freshsterile PBS (pre-incubated at 37° C.). Simultaneously, 50 μL (120 mg/mL)of mAb was injected in the VH-compartment. Devices were sealed andincubated at 37° C. for the defined time intervals i.e., initial,week-2, week-4 and week-8. The VH-compartment containing pVH or aVH,without mAb, were considered as negative controls. Also, to investigatethe effect of pH, temperature, and PBS on the stability of the mAb, theVH-compartment, filled with PBS, containing 50 μL of mAb was used ascontrol. At defined time intervals, samples were aseptically transferredfrom VH-compartments in sterile glass vials. The performance of themodel was evaluated by estimating the pH, osmolality and total proteinconcentration of samples. Samples were further evaluated to investigatephysical stability and binding affinity of mAb.

Experimental Setup of aVH in Vials

2 mL of sterile filtered aVH was aseptically transferred in the sterile6 mL glass vials (FIG. 3b ). 25 μL of the 120 mg/mL of mAb formulationwas injected in the test samples where as aVH without mAb was consideredas control. Vials were incubated at 37° C. and samples were collected atdefined time intervals i.e., initial, week-2, week-4 and week-8. Sampleswere analyzed by various analytical techniques to evaluate stability ofmAb.

TABLE 10 Study design to investigate mAb stability in natural VH andartificial VH in two different model i.e., ExVit and vial. ExVit ModelVH-compartment Buffer-compartment Vial Test-A aVH + mAb PBS — Test-BpVH + mAb PBS — Test-C — — aVH + mAb Control-A PBS + mAb PBS — Control-BaVH PBS — Control-C pVH PBS — Control-D — — aVH

Example 8

Stability of mAb in the aVH and Natural VH (Evaluation of ModelPerformance)

It was important to confirm that aVH can be investigated under ExVit andVial model in the presence of mAb. In order to confirm that Test groupsA, B & C and Control groups A, B, C and D (according to Table 10) wereinvestigated for changes in pH, osmolality and clarity following longterm incubation at 37° C. The pH was investigated at each time interval.Briefly, 100 μL of the samples were collected from the ExVit and vialmodel, from the controls (pVH alone, aVH alone, and buffer+mAb) and testarticles (pVH+mAb, aVH+mAb). The pH and osmolality were analyzed asdescribed earlier. The pH and osmolality results were plotted for thetime interval vs pH unit or Osmol/kg, respectively.

In-vivo, the VH is always buffered to physiological pH. Therefore, inorder to mimic the long-term stability of mAb, it is very important tomaintain the pH of the aVH or natural VH to physiological value. It canbe clearly depicted from the data shown in FIG. 4 a, that the pH valueof all the test and control samples (ExVit or Vial model) followingaddition of a mAb formulation remained stable throughout the studyperiod of 2 months. Furthermore, during the entire study, osmolality ofthe aVH and pVH were maintained between 0.31 and 0.33 Osmol/kg (FIG. 4b). It indicates that the mAb formulation was always homogeneouslydistributed within the aVH/pVH without altering their osmolality. Inthis study, aVH was found stable in the ExVit model as well as in thevial and hence protein stability investigated in the aVH can be comparedwith isolated pVH.

Example 9

Evaluation of Physical Stability of mAb in the aVH and Natural VH

Samples were evaluated for the physical stability of mAb by variousanalytical techniques e.g., microscopy, turbidimetry, size exclusionchromatography (SEC), and capillary electrophoresis sodium dodecylsulfate-non gel sieving (CE-SDS-NGS) as indicated in the following.

Microscopic Evaluation of Insoluble Particles

Proteins are generally very sensitive towards the physical, chemical andenvironmental stress which can easily generate soluble and insolubleparticles (large aggregates). To evaluate insoluble particles, at eachtime intervals 2 mL of the sample was transferred aseptically in sterileFTU tubes. The samples were assessed microscopically (Keyence VHX-600digital microscope) for the presence of visible particles.

Turbidimetry

Physical stability of protein could also be estimated by theturbidimetric analysis. Control and test samples at the selected timepoints were estimated for turbidity using 2100AN Turbidimeter (HACH).Instrument was calibrated between 0.1 and 150 NTU. To evaluateturbidity, at each time intervals 2 mL of sample was asepticallytransferred in the sterile FTU tubes. The results were plotted for FTUunit vs time (FIG. 5).

Size Exclusion Chromatography (SEC)

The level of fragments and soluble aggregates of mAb were assessed usingsize-exclusion chromatography (SEC) coupled with a UV-visible detector.The SEC method was developed in-house for the estimation of mAb.Briefly, 50 μL of samples (controls and test specimens) were injectedinto the separation column procured from Tosch Bioscience (TSK gel,G3000SWXL, 7.8-300 mm, 5μ). The separation of aggregates and fragmentswere carried out at a flow rate of 0.5 mL/min using 0.2 M phosphatebuffer (pH 7.0) as a mobile phase. UV-visible detection was performed at280 nm with a Waters-2489 detector (Water Corp. MA, USA). Analysis wasexecuted on a Waters 2695 HPLC (Waters Corp. MA, USA) and the data wasprocessed utilizing Empower-2 software.

Capillary Electrophoresis Sodium Dodecyl Sulfate-Non Gel Sieving(CE-SDS-NGS)

Levels of soluble aggregates and fragments in the stability samples werefurther confirmed by the CE-SDS-NGS. CE-SDS-NGS analysis was performedunder non-reducing condition with the Beckman Coulter CapillaryElectrophoresis System (Proteome Lab PA800). The capillary was rinsed at70 psi with 0.1 mM NaOH (for 5 min), 0.1 mM HCl (for 1 min) anddeionized water (for 1 min). The SDS MW gel buffer was loaded in thecapillary at 50 psi. Non-reduced samples were then injectedelectrokinetically at 10 kV and analysis was carried out at 15 kV. Thedata was processed using 32-Karat software. The area of the main peak,aggregates, fragments, light-chain and heavy-chain were calculated, andthe results were plotted as peak area (%) vs time.

Formation of particles and insoluble aggregates in test and controlsamples were assessed by observing the presence of visible particlesusing microscopic analysis (Microscopic evaluation and turbidity). Theresults exhibited no visible particles in any of the test or controlgroups after 2 months of incubation at 37° C. Also, no immediateprecipitation or particle formation was observed when 50 μL of the mAbformulation was injected into the test or controls. The results werefurther confirmed by the turbidimetric analysis (FIG. 5), whereturbidity readings for test groups remained below 10 FTU and they werealso comparable with the controls. It suggests that no visible orsub-visible aggregates were generated during the incubation of mAb inthe aVH.

Formation of soluble aggregates, although not experimentally proven, mayhave an impact on the safety and/or efficacy of protein drug. SECresults in FIGS. 6a and 6b demonstrate no change in the monomer contentof mAb following 2 months of incubation in the Test-B and C. This is aninteresting finding suggesting that mAb does not generate high molecularweight species (HMWS, soluble aggregates) and low molecular weightspecies (LMWS, fragments) in the pVH (Test-B) and also in the aVH whenincubated in the vial (Test-C). On the other hand, significant loss inthe main peak (2.3%) was observed when mAb was incubated in the PBS(Control-A) at 37° C. for the same amount of time. Surprisingly, Test-Ashowed minor loss in main peak at 2 month time point when compared withTest-C. In Test-C (Vial model), mAb is exposed only to the PBS presentin aVH as buffer system, whereas in Test-A (ExVit model) mAb is exposedadditionally to the PBS present in buffer-compartment as well. This mayexplain marginally higher loss in main peak observed in Test-A comparedto Test-C.

CE-SDS-NGS analysis was performed to evaluate physical stability and toassess mainly fragmentation of mAb after incubation in the PBS and VH at37° C. Results depicted in FIG. 7, demonstrate the loss of a main peakarea (FIG. 7a ) and concurrent increase in smaller molecular weightdeglycosylated variants (FIG. 7b ). Loss of the main peak was ca. 25% inthe Control-A group which was ca. 400% higher than the loss observed inthe Test-B (ca. 7%). Moreover, the area of the LMWS for the controlsample was increased more than 300% compared to the Test-B. Similarly asobserved in the SEC, mAb incubated in the aVH with ExVit setup (Test-A)showed higher loss in the main peak and increase in the LMWS compared tothe mAb incubated in the aVH with vial setup (Test-C). Moreover,physical stability of mAb in Test-A resembles more to the Control-A andphysical stability of mAb in Test-C aligns more to the Test-B. Itfurther confirms the results observed during SEC analysis. It isimportant to note that the LMWS observed in the Test-A, Test-B andTest-C are of the similar type which further supports the argument ofusing aVH in place of natural VH to predict in-vivo protein stability.

Example 10

Evaluation of Binding Affinity of mAb in the aVH and Natural VH

The specific interaction of mAb stability samples with their targetantigens was assessed using a SPR-Biacore T100/T200 instrument (GEHealthcare). The dual binding SPR assay was conducted to evaluate theinteractions between the mAb and its target antigens. The SPR assay wasperformed according to European Pharmacopoeia, Osmolality, Section2.2.35, European Directorate for the Quality of Medicine, Strasbourg,France, 2017, pp. 59, which is herein incorporated by reference in itsentirety. Capturing anti-human Fab antibody (Human Fab Capture Kit, GEHealthcare) was immobilized on a Biacore CM5-biosensor chip via standardamine coupling to achieve a coupling density of >5000 RU. Analysis wasconducted at 25° C. and a flow rate of 10 μL/min using PBS-T (phosphatebuffered saline containing 0.05% polysorbate 20, pH 7.4) as running anddilution buffer. The mAb stability samples (extracted from aVH/pVH/PBS)were injected on the measurement cell at various concentrations(13.85/19.04/26.18/36.00 μg/mL) for 90 s. Antigen-1 was injected for 60s at a concentration of 0.5 μg/mL followed by an injection of 2.5 μg/mLAntigen-2 for 60 s on both (measurement- and reference-) flow cells,complex stability of both antigens was monitored for 30 s. Afterwards,the chip surface was regenerated by a 60 s injection of 10 mM pH 2.1glycine solution at a flow rate of 30 μg/mL. Relative binding levels ofthe antigens to captured mAb samples were adjusted by normalization ofthe mAb sample capture level relative to the reference material with adefined activity of 100%. The obtained relative activity of theindividual binding domains to their respective antigens was plottedagainst the incubation time.

As shown in FIGS. 8a and 8 b, a significant loss in the binding affinityof mAb to antigen-1 (20% loss) and antigen-2 (10% loss), respectivelywas observed in Test groups A, B and C. Though there was a differenceobserved in the physical stability of mAb in Test groups A, B and C, theloss in the binding affinity was not significantly different betweenthese groups. It may be due to the fact that chemical alteration in theCDR of the antibody has direct impact on its binding affinity towardsthe antigen. The chemical stability of this mAb is highly impacted byenvironmental factors such as pH and temperature. In the stabilitystudy, pH and temperature were constant in all the groups throughout thestudy period. Hence, chemical stability of mAb in all the groups mightget impacted equally and thus binding affinity of the mAb was alsoimpacted similarly.

Example 11

Preparation of Small Molecule (SM) Active Pharmaceutical Ingredient(API)-Loaded PLGA Implants and In-Vitro Release of API From Long-ActingDelivery (LAD) Implants in the aVH

These LAD implants are produced by hot-melt extrusion process, verysimilarly to the marketed product Ozurdex which is comprised ofDexamethasone as SM API and PLGA as matrix forming polymer. TheSM-loaded poly(lactic-co-glycolic acid (PLGA) implants were preparedusing already established in house hot-melt extrusion protocol. Briefly,four different implants (F-1, F-2, F-3 and F-4) were prepared usinghot-melt extrusion. These implants were varying in polymer type(hydrophobicity and end cap), polymer molecular weight, and extrusiontemperature. Detailed composition and process parameters of implants aredepicted in Table 11. To prepare implants, polymer and API werehomogenously mixed and extruded at two different temperatures (F-1 andF-2 at 90° C., and F-3 at 110° C.). Further, to understand the impact ofsterilization on the quality of implant, a part of F-3 batch wassterilized (F-4) using E-beam sterilization method. E-beam sterilizationwas performed at a standard target dose of 25kGy, at room temperatureusing simultaneous beam processing 10 MeV, 20 KW linear acceleratorsMevex (Ottawa, CN). All the implants were then immediately stored at2-8° C. until further use.

TABLE 11 Composition and process parameters of SM-loaded PLGA LADimplants D,L-Lactide/ Glycolide End MW % Drug Extrusion Implants RatioGroup (kDa) Loading temp. Sterilization F-1 75:25 Ester 100 25 90 No F-275:25 Acid 30 25 90 No F-3 75:25 Ester 100 25 110 No F-4 75:25 Ester 10025 110 Yes (E-Beam)

These implants were characterized for various key parameters such asquantity, homogeneity and stability of API following extrusion, andimportantly for release of API. In-vitro and in-vivo release of API fromLAD implants can be easily impacted by complex interdependentformulation and process variables. These parameters could be related tomatrix forming polymer (such as MW of polymer, hydrophobicity ofpolymer, etc.), and/or API (hydrophobicity, physical state of API suchas polymorph or salt forms), and/or related to processes (such assterilization, extrusion temperature, etc.). To capture the impact ofthese variables on the release of API, it is very important to selectthe correct release medium. Therefore, the possibility of aVH to be usedas a release medium for the LAD implants intended for intravitrealadministration was evaluated. Briefly, a 5 mg of above differentimplants (F-1 to F-4) were incubated in 2 mL of an aVH for 4 weeks at37° C. Samples were collected after 1, 7, 14, 21 and 28 days ofincubation, and analyzed by HPLC method (developed in house) for thequalitative and quantitative estimation of SM. As depicted in FIG. 9,all the implants showed different levels of SM release after 4 weeks.Implant F-2 was comprised of fastest degrading polymer and hence itshowed the fastest release compared to all other implants. Implants F-1and F-3 were identical in composition but extruded at differenttemperature. F-3 exhibited significantly higher release compared to F-1.This difference in release might be attributed to the fact that F-3 isextruded above the Tg (96° C.) of API. The higher extrusion temperaturemay have resulted in homogeneous blending of hydrophobic drug withpolymer which eventually leads to faster release of API. Whereas F-2 wasextruded below the Tg of API and therefore API may have remained in itsoriginal particulate form leading to non-homogeneous mixing withpolymer. Because API is hydrophobic, these API particles might haveexhibited very poor dissolution and diffusion based release in an aVH.Interestingly, F-4 showed significantly slower release compared to F-3.It might possible that the process of e-beam sterilization hasintroduced structural changes in the biodegradable PLGA polymer whichhas resulted in slower release of API.

It is important to note that aVH as a release medium was able todifferentiate the implants based on their key quality feature i.e.,release of API. This experiment in aVH clearly enables to seedifferences between different types of polymers and also capturesdifferences coming from processing such as temperature andsterilization. In other words, aVH as release medium is able todiscriminate between formulations that are known to, or at leastexpected to perform differently. Hence, aVH can not only be used toinvestigate the stability of formulations (as highlighted in previousexamples) but also can be used to investigate release of API from LADformulations. Furthermore, aVH as disclosed herein can better mimic thein vivo conditions with regard to API release and/or drug release fromLAD implants.

1. An artificial vitreous humor composition comprising a phosphatebuffer, wherein the phosphate buffer has a pH value in the range from7.0 to 7.7.
 2. The artificial vitreous humor composition of claim 1,wherein the phosphate buffer is phosphate buffer saline (PBS) in therange from 0.001 to 0.2 M.
 3. The artificial vitreous humor compositionof claim 1, wherein the composition comprises at least one of up to 578μM creatinine, up to 28.1 mM glucose, up to 58.5 mM urea, 200 to 1630 μMxanthine, 100 to 800 μM hypoxanthine, 0.1 to 23 mM sodium lactate, and50 to 300 μM glutathione.
 4. The artificial vitreous humor compositionof claim 1, wherein the composition comprises 64.6 μM creatinine, 2.2 mMglucose, 7.6 mM urea, 580 μM xanthine, 309 μM hypoxanthine, 10.5 mMsodium lactate and 200 μM glutathione.
 5. The artificial vitreous humorcomposition of claim 1, wherein the composition comprises 20 to 300 mg/Ltype II collagen.
 6. The artificial vitreous humor composition of claim1, wherein the composition does not comprise ascorbic acid.
 7. A methodfor the production of an artificial vitreous humor composition,comprising the steps (i) Providing stock solutions of glucose,creatinine, sodium lactate, glutathione and urea in water, ofhypoxanthine in formic acid:water of 2:1 ratio, and of xanthine sodiumin 1M NaOH; (ii) Mixing of stock solutions of step (i) in a phosphatebuffer with a pH value in the range from 7.0 to 7.7, and pH adjustmentto the pH value of the phosphate buffer; (iii) Addition of hyaluronicacid to the mixture obtained in step (iii) and stirring at 2 to 8° C. atleast for 4 hours; (iv) Addition of collagen type-II to the mixtureobtained in step (iii); (v) Optionally sterile-filtering of the mixtureobtained in step (iv).
 8. A method for analyzing the behavior of asubstance comprising the steps (i) providing an artificial vitreoushumor composition of claim 1 in an in-vitro environment; (ii) applyingthe substance to the artificial vitreous humor; and (iii) determining atleast one property of the applied substance.
 9. The method of claim 8,wherein the substance to be applied is at least one of a macromolecule,a drug formulation, an excipient, a protein or a combination thereof.10. The method of claim 8, wherein the substance to be applied comprisesmolecules having a size in a range between 100 Da and 1800 kDa.
 11. Themethod of claim 8, wherein the at least one property of the appliedsubstance is selected from the group consisting of stability,bioavailability, release from DDS system and degradation of DDS system.12. The method of claim 8, wherein the applied substance is left in theartificial vitreous humor composition for up to 360 days, prior to step(iii).
 13. A method for analyzing the behavior of an artificial humorcomposition comprising the steps (i) providing an artificial vitreoushumor composition of claim 1 in an in-vitro environment; (ii) applying asubstance to the artificial vitreous humor; and (iii) determining atleast one property of the artificial humor composition.
 14. Anartificial vitreous humor composition produced by the method of claim 7.15. A kit of parts for the production of an artificial vitreous humorcomposition, comprising (A) a phosphate buffer, having a pH value in therange from 7.0 to 7.7; (B) at least one selected from the groupconsisting of creatinine, glucose, urea, xanthine, hypoxanthine, sodiumlactate, glutathione; (C) type II collagen and/or sodium hyaluronate;and optionally (D) instructions for use.
 16. (canceled)
 17. Use of aphosphate buffer saline having a pH value in the range from 7.0 to 7.7for the production of an artificial vitreous humor composition accordingto claim
 1. 18. (canceled)